The underlying hypothesis is that internal electric fields in heme proteins influence the protein stability and reactivity of the heme. Three specific aims are proposed. The first is to examine the nature of surface of proteins with an emphasis on water interactions. Infrared spectroscopy of isotopically labeled peptides will answer questions of the role of neighboring amino acids in influencing amide-water H-bonds. Proteins will be examined in glasses, and single molecule detection used to isolate unfolded forms of the protein. The second goal is to predict the effect of heme environment upon optical and IR spectra. Features in the infrared and visible spectra of horseradish peroxidase and cytochrome c will be determined and then interpreted in light of continuum electrostatics, molecular dynamics and quantum mechanic calculations. The intention is to understand how the protein modulates the characteristics of particular groups. The third aim is the quantitative characterization of the dynamics of proteins and the role of dynamics on internal reactions. An electron transfer reaction between bound substrate and excited state porphyrin in horseradish peroxidase will be examined as a function of solvent viscosity and temperature. Solvent dependent and independent motions will be separated, and the results will be correlated with molecular mechanics. Additionally, we ask about the role of dynamics in allowing the diffusion of oxygen through the polypeptide chain of horseradish peroxidase. Using a phosphorescence quenching method, the oxygen accessibility will be studied. Peroxidase has a channel to the heme reachable to the solvent. The x-ray structure shows that adding substrate blocks this channel, with little change in protein conformation. It follows that if oxygen accessibility to the heme occurs mainly through the channel, then the oxygen quenching reaction will be inhibited by the substrate.